muramidase and 1-2-azaborine

A congeneric series of 1,2-azaborine ligands was used to study the relationship between the steric demand of the ligand and hydrogen bonding strength in the context of ligand-protein binding using engineered T4 lysozymes as the model biomacromolecules. The hydrogen bonding strength values were extracted from experimentally accessible binding free energies using the Double Mutant Cycle analysis. With the increasing steric demand, the NH…102Q hydrogen bonding interaction is weakened; however, this weakening of the hydrogen bonding interaction occurs in discrete steps rather than in an incremental fashion.

Topics: Bacteriophage T4; Boron Compounds; Hydrogen Bonding; Ligands; Molecular Structure; Muramidase; Protein Engineering; Thermodynamics

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

Topics: Bacteriophage T4; Boron Compounds; Chemistry Techniques, Synthetic; Diffusion; Muramidase; Mutation; Proteins

Protein crystallography and calorimetry were used to characterize the binding of 1,2-azaborines to model cavities in T4 lysozyme in direct comparison to their carbonaceous counterparts. In the apolar L99A cavity, affinity for Ab dropped only slightly versus benzene. In the cavity designed to accommodate a single hydrogen bond (L99A/M102Q), Gln102═O···H-N hydrogen bonding for Ab and BEtAb was observed in the crystallographic complexes. The strength of the hydrogen bonding was estimated as 0.94 and 0.64 kcal/mol for Ab and BEtAb, respectively. This work unambiguously demonstrates that 1,2-azaborines can be readily accommodated in classic aryl recognition pockets and establishes one of 1,2-azaborine's distinguishing features from its carbonaceous isostere benzene: its ability to serve as an NH hydrogen bond donor in a biological setting.

Topics: Boron Compounds; Calorimetry; Hydrogen Bonding; Models, Molecular; Molecular Structure; Muramidase; Thermodynamics